Modulation of protein /cell functions by heparin/heparan

肝素/乙酰肝素调节蛋白质/细胞功能

• 批准号: 6992842
• 负责人: AUDREY L STONE
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6992842
• 关键词: antiAIDS agent; antimalarial agents; bacterial polysaccharides; biomimetics; carbohydrate structure; chemical models; chemical structure function; chromatography; combinatorial chemistry; conformation; drug design /synthesis /production; heparan sulfate; heparin; infrared spectrometry; nuclear magnetic resonance spectroscopy; oligosaccharides; virus infection mechanism

This project elucidates the fundamental molecular design of the heparin/ heparan sulfate (H/HS) class of biological regulatory polysaccharides and studies how the high degree of diversity of its sulfated oligosaccharide (oligoS) structures relates to its parallel, multifunctional capacity to modulate the functional activity of numerous diverse protein partners in normal and disease processes (e.g., cell growth, cancer, secretion, multi-cell reactions in development, blood coagulation, physiological stability, and in infections by virus and other classes of human pathogens). Within this basic research map, the project pursues the development of H/HS-mimetic therapeutic agents against various disorders and pathogens (in particular HIV-1), based on the known glycobiology of the multifunctional family of H/HS and an H/HS-mimetic pharmaceutical which is comprised of a mixture of sulfated xylan oligocaccharides (S-oligoS) and closely mimics most of the biological actions of heparin. Newly devised and/or established biological, biochemical, and physical methods are utilized. Due to the complex diversity of sequences within H/HS chains, libraries of various unique H/HS oligoS have not been available for extended research. WE PREVIOUSLY devised an approach to overcome this roadblock. A family of H/HS-mimetic S-oligoS (a 24-Component library) was produced by fractionation of the pharmaceutical, thus generating a macro combinatorial strategy. This enabled us to examine whether a given heparin-mimetic function in vitro, like those of heparin, was governed by a degree of structural specificity within the family, and whether the active Component would likely lack bleeding toxicity as well. Both these characteristics are required indicators of usefulness for further drug development. We engaged several approaches to study the structure/function relations of the H/HS-mimetic inhibition of HIV-1. Early findings showed that Components of our H/HS-mimetic library displayed differential capacities to inhibit the cytotoxicity or the syncytium-forming toxicity of HIV-1, demonstrating that the inhibitions were governed by a degree of structural specificity. This supported our hypothesis that the anti-viral capacities of H/HS-mimetic S-oligoS follow rules of specificity, similar to those of H/HS and led us to the development of CpF-PkII, a highly active, anti-HIV-1 S-oligoS which was essentially free of anti-thrombin toxicity. Enlargement of preparative methods was devised within the limitations of the 400 mg/load imposed by the soft gel nature of the fractionating matrix, and a clinical preparation utilizing 40 grams of starting pharmaceutical has been underway to obtain sufficient agent for Phase I and other potential preliminary trials. THIS YEAR, further analysis of the structure/function relations of our S-oligoS library, including findings from studies on additional H/HS-modulating functions, showed that they are consistent with the known molecular glycobiology of H/HS, derived from the now classical model of antithrombin regulation of serine esterase enzymes in the clotting cascade, first discovered by Robert D. Rosenberg. Here, activation of a single inhibitory protein is multifunctional and two types of binding may occur between H/HS and antithrombin. One involves the oligoS sequence which provides most of the specific binding strength and effectuates an activating conformational change in antithrombin against the given enzyme. The second is nuanced, involving in the case of thrombin for example, both the specific oligoS binding sequence plus an additional binding of another specific H/HS sequence, which is required to fully activate the protein against thrombin. Our CUMULATIVE FINDINGS have broad significance in laying to rest a long outdated bias that the heparin-like effects of sulfated but structurally unrelated polysaccharides must be non-specific and due to the high sulfate charge density. Below, we show also that the structure of the native polysaccharide dose not provide a valid clue about the structure of the active oligoS. THIS YEAR, we completed phase I fractionation of starting material and rechromatography to the CpF-PkII.stage is continuing; biological and physicochemical analyses are continuing. This putative drug would PROVIDE A SIGNIFICANT ADDITIONAL MECHANISM OF INHIBITION OF HIV-1 since its mode of action in vitro prevents the binding and entry of virus into target cells and the cell-cell spreading of the infection. Peak II also displays a moderate inhibitory capacity against the merozoite invasion of RBC by malarial parasites in vitro. Additional library Components are underway for studies on other potential therapeutic uses, e.g., the various anti-malarial capacities of the S-oligoS. Elucidation of the S-OLIGOS STRUCTURE by biochemical, chemical, and spectroscopic (FTIR, NMR, and dye-coupling) analyses revealed previously for the first time that the H/HS-mimetic S-oligoS were similar and significantly different from the primary structure of the native polyxylan which has one monomeric GlcA branch per 10 Xyl on the average. The primary structure of the S-oligoS contained a tetraS motif, one GlcA branch per 3 Xyl on average. This could generate a sulfated GlcA-xyl Di-S spaced by two xylose moieties along the backbone, each GlcA assuming a helical rotation of 180 degrees. Furthermore, FTIR and heteronuclear two-dimensional NMR proton-13C-correlation (HMQC) spectroscopy revealed for the first time that CpF-PkII contained some sugar moieties in an alternative chair conformation instead of the expected normal chair form; such sugars would have a shortening effect on the chain backbone. Further FTIR data indicated that these alternate sugar conformations are present in other H/HS-mimetic Components but that the ratio of normal to alternative forms differs among the various S-oligo. That H/HS-mimetic S-oligoS contain a tetraS motif involving GlcA in their back bone repeat and that their sugar conformations may be a governing factor in structural specificity of the protein-binding sugar sequences has SIGNIFICANT IMPORTANCE, e.g., in strategies for synthesis of oligoS drugs and the design of synthetic oligoS-protective antigens. It is of note that, more recently, the anticoagulant capacity of the synthetic antithrombin-binding pentaS was shown to reside uniquely in the form containing an alternative skewed-ring conformation in the iduronic acid moiety. THIS YEAR preparation of library Components for further studies on these structure/function relations have continued. Manuscripts in preparation: 1. Structure-function relations of a library of heparin-mimetc sulfated xylan oligosaccharides: Preparation of a potent HIV-1 inhibitor and structural characterization by FTIR and NMR spectroscopy and glucoronic acid analysis, by Audrey Larack Stone, Alexis F. Henry, Derek J. Melton, James McMahon and Maria O. Longas (Bioploymers); 2. Structure-function relations of a library of heparin-mimetic sulfated xylan oligosaccharides: Study of alternative chair sugar conformation by heteronuclear two-dimensional NMR 1H-13C-correlation spectroscopy and HMQC analysis, Maria O. Longas and Audrey Larack Stone (J. Biomedicine and Biotechnology); 3. Structure-Function relations of heparins/heparan sulfates and heparin-mimetic sulfated xylan oligosaccharides: A role of secondary structure in functional specificity as viewed from asymmetric metachromatic coupling reactions, by Audrey Larack Stone (Macromolecular Chemie).

This project elucidates the fundamental molecular design of the heparin/ heparan sulfate (H/HS) class of biological regulatory polysaccharides and studies how the high degree of diversity of its sulfated oligosaccharide (oligoS) structures relates to its parallel, multifunctional capacity to modulate the functional activity of numerous diverse protein partners in normal and disease processes (e.g., cell growth, cancer, secretion,多细胞在发育，血液凝结，生理稳定性以及病毒和其他类型人类病原体感染中的反应。在这张基础研究图中，该项目基于已知的H/HS多功能家族的糖生物学以及H/HS-MIMETIC PHARMATAICATIC的多功能糖生物学，追求针对各种疾病和病原体（尤其是HIV-1）（尤其是HIV-1）（尤其是HIV-1）的项目开发的。肝素的作用。使用新设计和/或已建立的生物学，生化和物理方法。由于H/HS链中序列的复杂多样性，因此尚未用于扩展研究的各种独特的H/HS寡聚的库。我们以前设计了一种克服这一障碍的方法。 H/hs-Mimetic S-Oligos（一个24组分库）的家族是通过药物分馏而产生的，从而产生了宏观组合策略。这使我们能够检查给定的肝素模拟功能在体外（例如肝素的功能）是否受家庭内部的结构特异性的影响，以及活性成分是否也可能缺乏出血毒性。这两种特征都是为进一步的药物开发的有用性指标。我们参与了几种研究H/HS模拟抑制HIV-1的结构/功能关系的方法。早期发现表明，我们的H/HS模拟文库的组成部分显示出差异能力以抑制HIV-1的细胞毒性或合成形成毒性的毒性，表明抑制作用受一定程度的结构特异性控制。这支持了我们的假设，即H/HS-Mimetic S-Oligos的抗病毒能力遵循特异性规则，类似于H/HS的规则，并导致我们发展了CPF-PKII的发展，CPF-PKII是一种高度活跃的抗HIV-1 S-Oligos，这基本上没有抗thrombin毒性。制备方法的扩大是在分级矩阵的软凝胶性质施加的400 mg/载荷的局限性中，并利用40克开始药物的临床制备正在为I期和其他潜在的预初次试验提供足够的药物。 THIS YEAR, further analysis of the structure/function relations of our S-oligoS library, including findings from studies on additional H/HS-modulating functions, showed that they are consistent with the known molecular glycobiology of H/HS, derived from the now classical model of antithrombin regulation of serine esterase enzymes in the clotting cascade, first discovered by Robert D. Rosenberg.在这里，单个抑制蛋白的激活是多功能的，H/HS和抗凝血酶之间可能发生两种类型的结合。其中一种涉及寡素序列，该序列提供了大多数特定的结合强度，并影响抗凝血酶与给定酶的激活构象变化。第二个是细微的，例如在凝血酶的情况下，特定的寡寡核体结合序列以及另一个特定的H/HS序列的附加结合，这是完全激活蛋白质针对凝血酶的蛋白质所必需的。我们的累积发现在铺设长期过时的偏见方面具有广泛的意义，即硫酸盐但无关的多糖的肝素样作用必须非特异性，并且由于硫酸盐电荷密度很高。在下面，我们还显示天然多糖剂量的结构没有提供有关活性寡糖结构的有效线索。今年，我们完成了第一阶段的起始材料和复古分数的分数，阶段仍在继续。生物学和物理化学分析仍在继续。这种推定的药物将提供抑制HIV-1的重要附加机制，因为其体外作用方式可防止病毒进入靶细胞和感染的细胞细胞扩散。 Peak II还显示出对疟疾寄生虫在体外对RBC侵袭的中等抑制能力。正在进行有关其他潜在治疗用途的研究，例如S-Oligos的各种抗疟疾能力。 先前首次通过生化，化学和光谱（FTIR，NMR和染料耦合）分析来阐明S-橄榄石结构（FTIR，NMR和染料耦合）分析，首次揭示了H/HS-MIMIMETIC S-OLIGOS与本地聚二甲烷的主要结构相似，并且具有单一glca glca分支均为平均分支机构的原始聚合物的主要结构显着且显着差异。 S-Oligos的主要结构包含一个Tetras基序，平均每3 xyl一个GLCA分支。这可能会产生一个由两个木糖部分沿主链分布的硫酸化GLCA-xyl二二，每个GLCA都假设螺旋旋转180度。此外，FTIR和异核二维NMR Proton-13c - 相关（HMQC）光谱首次揭示了CPF-PKII在替代椅子构象中含有一些糖基部分，而不是预期的正常椅子。这样的糖会对链条主链产生缩短作用。进一步的FTIR数据表明，在其他H/HS模拟成分中存在这些替代糖构象，但是在各个S-Oligo之间，正常形式与替代形式的比率有所不同。 H/HS模拟的S-Oligos包含涉及GLCA后骨重复的Tetras基序，并且它们的糖构象可能是蛋白质结合糖序列结构特异性的统治因素，例如，在Oligos药物合成的策略中，糖构型具有重要的重要性。值得注意的是，最近显示，合成抗凝血素结合的五角根的抗凝能力被证明以唯一的形式居住，该形式含有含有替代性偏斜构象的形式。今年为这些结构/功能关系的进一步研究的图书馆组件的准备持续了。 制备中的手稿：1。肝素 - 含量硫酸二甲烷基寡糖的结构功能关系：通过FTIR和NMR光谱和葡萄糖酸分析的有效HIV-1抑制剂和结构表征的制备（Bioploymers）； 2。肝素模拟硫化Xylan寡糖库的结构功能关系：通过异核二维NMR 1H-13C - 13C - 相交光谱和HMQC分析研究替代椅子糖构象的研究，Maria O. Longas和Audrey Larack Stone（J. Biomicicine and Audrey Larack Stone） 3。肝素/肝素硫酸盐和肝素模拟硫酸二氢寡糖的结构 - 功能关系：二级结构在功能特异性中的作用，如从不对称的载体偶联反应所看的，由Audrey Larack Stone（Macromomolomecular Chemie）所看的。